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Báo cáo " Culturing of Chlorella vulgaris - Standard Operating Procedure "

Chlorella vulgaris is a planktonic unicellular green alga used as a food source for Daphnia magna (may not be suitable for D. pulex as cells are very large – pers com with a number of people over the years). Thus, this SOP describes the culture methods used for C. vulgaris intended for D. magna. Chlorella vulgaris is cultured in Bold’s Basal Medium, prepared as described in SOP BBM at end of this document. | Daphnia Research group University of Reading Last updated July 2007 Culturing of Chlorella vulgaris - Standard Operating Procedure Introduction Chlorella vulgaris is a planktonic unicellular green alga used as a food source for Daphnia magna may not be suitable for D. pulex as cells are very large - pers com with a number of people over the years . Thus this SOP describes the culture methods used for C. vulgaris intended for D. magna. Chlorella vulgaris is cultured in Bold s Basal Medium prepared as described in SOP BBM at end of this document. Cultures are maintained in 5 L glass fermenter bottles and aerated 0.2 pm filtered to aid gas exchange and to keep algal cells in suspension. Precautions must be taken at all times when preparing the inoculum fermenter vessels and removing the algae to prevent microbial contamination. This involves use of sterile techniques at all times. The culture is maintained on a semi-continuous basis. This means that once a week when the cultures are established and sufficiently green at least 500 ml of the algal suspension is removed and the fermenter contents replenished with fresh BBM medium. This method of maintenance results in the density of algal cells in the culture fluctuating with time. The removed algal suspension is then used to prepare the D. magna food. Take algae from alternate fermenters keep at least two cultures running simultaneously every week or when necessary so as to allow enough time for re-growth. Record volumes taken from each vessel and volume of food prepared at OD 1 10 dilution 440nm - 10mm pathway in logbook. NB Do not keep fermenters active for more than three months as dead cells accumulate at the bottom of the vessel and will if fed to the daphnia cause gastric blockages. I.e. the food quality declines. If there is the slightest suspicion that a fermenter may be contaminated discard without hesitation. Algal Slopes Chlorella vulgaris is stored on agar slopes as a reserve and to inoculate future .